1. Field of the Invention
The present invention relates to immortalized female muntjac cell lines, a method for purifying nonmuntjac chromosomes utilizing such cell lines, and hybrid muntjac cell lines containing nonmuntjac chromosomes or chromosome fragments.
2. Discussion of the Background
Chromosome sorting is a powerful technique for gene mapping and isolation, but it has remained largely within the purview of few specialized centers. Furthermore, despite the successful generation of several highly purified chromosome-specific cosmid libraries (Deaven, L. L., Cold Spring Harbor Symposia on Quantitative Biology, Vol. L1, pp. 159-167 (1986); Fuscoe, J. C., Cytogenet. Cell Genet., Vol. 43, pp. 79-86 (1986); and Van Dilla, M. A., Cytometry, Vol. 11, pp. 208-218 (1990)), the ability to generate pure single chromosome fractions has generally been limited. Historically, rodent cell lines have been used for generating hybrid cell lines prior to chromosome purification, because of the relatively large number of mutant cell lines enabling selection (Ruddle, F. H., Ann. Rev. Genet., Vol. 9, pp. 407-486 (1981)). However, with few exceptions, rodent cell lines contain chromosomes of the approximate size of most donor chromosomes, thus requiring specialized techniques and limiting the purity of the resulting sorted chromosomes (Fuscoe, J. C., Cytogenet. Cell Genet., Vol. 43, pp. 79-86 (1986); and Van Dilla, M. A., Cytometry, Vol. 11, pp. 208-218 (1990)).
Thus, most of the libraries containing human chromosomes contain significant impurities. For example, a summary of the gene libraries for each chromosome available to the general public reveals that the hamster impurity in these libraries ranges from 11% to 55%, with an average impurity of 31.9% (Van Dilla, M. A., et al, Cytometry, vol. 11, pp. 208-218 (1990)).
Chromosome separations based on size have been attempted previously by zonal rotor centrifugation (Stubblefield, E., Cytometry, Vol. 2, pp. 273-281 (1991)), swinging bucket centrifugation (Mendelsohn, J., J. Mol. Biol., Vol. 32, pp. 101-112 (1968)), or velocity sedimentation in specialized chambers (Collard, J. G., Exp. Cell Res., Vol. 130, pp. 217-227 (1980)). However, separation of human chromosomes to homogeneity was not successful until the advent of flow sorting of human monochromosome hybrids in rodent cells. Even then, flow sorting has required the use of specialized techniques and equipment available only in a small number of centers, such as multi-dimensional sorting (Langlois, R. G., Proc. Natl. Acad. Sci. USA, Vol. 79, pp. 7876-7880 (1982)), pulse-shape flow cytometry (Bartholdi, M. F., Cytometry, Vol. 11, pp. 165-172 (1990)), fringe-scan (Mullikin, J., Cytometry, Vol. 9, pp. 111-120 (1988)), or slit-scan flow cytometry (Lucas, J. N., Cytometry, Vol. 8, pp. 273-279 (1987)), and computer aided analysis (van den Engh, G., Cytometry, Vol. 11, pp. 173-183 (1990)). With some exceptions, the resulting sorted chromosomes are still relatively impure, because of the similarity in size to one or more rodent chromosomes (Deaven, L. L., Cold Spring Harbor Symposia on Quantitative Biology, Vol. L1, pp. 159-167 (1986); Fuscoe, J. C., Cytogenet. Cell Genet., Vol. 43, pp. 79-86 (1986); and Van Dilla, M. A., Cytometry, Vol. 11, pp. 208-218 (1990)).
The Indian muntjac, a small Asian barking deer, has the fewest diploid chromosomes of all mammals (Wurster, D. H., Science, Vol. 168, pp. 1364-1366 (1970)), with only 6 chromosomes (1,2,X) in the female and 7 in the male (1,2,X,Y), and the single laser, bivariate flow sorting of Indian muntjac chromosomes has been reported (Levy, H. P., et al, Cytometry, vol. 12, pp. 695-700 (1991)). Indian muntjac cells have been transformed for tumorigenicity studies with murine sarcoma virus (MSV) (Yuasa, Y., Gann, Vol. 69, pp. 441-445 (1978); and Hatanaka, M., J. Expt. Med., Vol. 150, pp. 1195-1201 (1979)), avian sarcoma virus (ASV) (Yuasa, Y., Gann, Vol. 69, pp. 441-445 (1978)), and SV40(Yamaguchi, N., J. Gen. Virol., Vol. 42, pp. 289-296 (1979)), but long-term karyotypic stability was not established (Yuasa, Y., Gann, Vol. 69, pp. 441-445 (1978); Hatanaka, M., J. Expt. Med., Vol. 150, pp. 1195-1201 (1979); and Yamaguchi, N., J. Gen. Virol., Vol. 42, pp. 289-296 (1979)).
One immortalized male muntjac cell line has been reported (Pillidge, L., et al, Int. J. Radiat. Biol., vol. 50, pp. 119-136 (1986)). However, this cell line is so unstable that it is used as a model of karyotypic instability and defective DNA repair mechanisms (Pillidge, L., et al, Mutation Research, vol. 166, pp. 265-273 (1986); Bouffler, S. D., et al, Somatic Cell and Molecular Genetics, vol. 16, pp. 451-460 (1990); Musk, S. R. R., et al, Biochimica et Biophysica Acta, vol. 1052, pp. 53-62 (1990); Jha, B., et al, Mutation Research, vol. 254, pp. 13-25 (1991); Godfrey, D. B., et al, Mutation Research, vol. 274, pp. 225-235 (1992); and Musk, S. R. R., et al, Mutation Research, vol. 300, pp. 111-117 (1993)).
A readily available non-immortalized cell strain, from a male, contains a Y chromosome of comparable size to human group C chromosomes (American Type Culture Collection, Catalogue of Cell Lines and Hybridomas, Ed. 7 Rockville, Md.: American Type Culture Collection, p. 89 (1992)).